Abstract
In order to determine phenotypic protease and reverse transcriptase inhibitor-associated resistance in HIV subtype C virus, we have synthetically constructed an HIV-1 subtype C (HIV-1-C) viral backbone for use in a recombinant virus assay. The in silico designed viral genome was divided into 4 fragments, which were chemically synthesized and joined together by conventional subcloning. Subsequently, gag-protease-reverse-transcriptase (GPRT) fragments from 8 HIV-1 subtype C-infected patient samples were RT-PCR-amplified and cloned into the HIV-1-C backbone (deleted for GPRT) using In-Fusion reagents. Recombinant viruses (1 to 5 per patient sample) were produced in MT4-eGFP cells where cyto-pathogenic effect (CPE), p24 and Viral Load (VL) were monitored. The resulting HIV-1-C recombinant virus stocks (RVS) were added to MT4-eGFP cells in the presence of serial dilutions of antiretroviral drugs (PI, NNRTI, NRTI) to determine the fold-change in IC50 compared to the IC50 of wild-type HIV-1 virus. Additionally, viral RNA was extracted from the HIV-1-C RVS and the amplified GPRT products were used to generate recombinant virus in a subtype B backbone. Phenotypic resistance profiles in a subtype B and subtype C backbone were compared. The following observations were made: i) functional, infectious HIV-1 subtype C viruses were generated, confirmed by VL and p24 measurements; ii) their rate of infection was slower than viruses generated in the subtype B backbone; iii) they did not produce clear CPE in MT4 cells; and iv) drug resistance profiles generated in both backbones were very similar, including re-sensitizing effects like M184V on AZT.
Highlights
Subtype C of the Human Immunodeficiency Virus type 1 (HIV-1) is accountable for over 50% of the HIV-1 infections worldwide [1,2,3,4]
If the samples were processed for viral load determination on the EasyQ, EasyQ calibrator was added along with the magnetic silica according to the guidelines of the manufacturer
A total of 8 colonies were picked per In-Fusion reaction (1 In-Fusion/ amplicon) and sequenced, to verify a correct insert of the GPRT
Summary
Subtype C of the Human Immunodeficiency Virus type 1 (HIV-1) is accountable for over 50% of the HIV-1 infections worldwide [1,2,3,4]. An adequate resistance-profiling tool requires an assay that correctly assesses drug resistance for all HIV variants. This can be a challenge as even quasi-species in a single individual may differ up to 10% [2]. In order to generate correct sensitive/ resistant calls, the sequence interpretation algorithm needs to be able to integrate the constantly growing knowledge of resistanceassociated mutations (RAMs). This should include different possible pathways to anti-retroviral drug resistance among the different subtypes
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